This application proposes to study (1) the role of alanine uptake by liver as a rate-influencing step in gluconeogenesis from alanine, (2) the importance of mitochondrial transport and metabolism of alanine in the pathway to glucose synthesis, and (3) the effect of diabetes mellitus and other states of altered gluconeogenesis on hepatic alanine uptake. Studies in vivo and in vitro will be performed to accomplish the first objective. In studies with dogs, the portal venous plasma concentration and radiospecific activity of alanine will be controlled at a constant level and manipulated at will to determine the relationships of portal venous and intrahepatic alanine concentrations and rate of conversion of alanine to glucose in the fed (control) and fasted states and following administration of glucagon. These relationships will be further explored in studies using perifused isolated hepatocytes. Alpha-Amino-isobutyric acid, an inhibitor of alanine transport in liver, will be used in studies in vivo and in vitro to inhibit hepatic alanine transport and to define the role of this inhibition in gluconeogenesis from alanine. The second objective will be accomplished by use of isolated hepatocytes and mitochondria and inhibitors of mitochondrial pyruvate transport. The aims will be to characterize the role of intramitochondrial transamination of alanine in gluconeogenesis and the properties and regulation of mitochondrial alanine transport. The third objective will be accomplished by the use of isolated hepatocytes and mitochondria prepared from animals in the refed state, with experimentally induced diabetes mellitus, and animals with augmented gluconeogenesis by treatment with phloridzin.